1. Field of the Invention
This invention generally relates to a spectrophotometric method of quantitatively determining the concentration of a dilute component in a light- or other radiation-scattering environment containing the dilute component in combination with a reference component of known concentration.
2. Description of the Related Art
In many fields of technology there is a need for quantitative determination of dilute component concentrations in environments where the dilute component is in combination with a reference component of known concentration. Examples of illustrative environments of such type include enzymes, proteins, and metabolites in corporeal fluids; acidic fumes or gaseous components (e.g., hydrogen sulfide and sulfuric acid, nitric acid, carbon monoxide, etc.) in the atmosphere; salt concentrations in sea water undergoing desalination; ozone in ozone-enriched air utilized in waste water ozonation systems, etc.
In particular, there has been a specific need in the medical and health care fields for a non-invasive, continuous, atraumatic, in vivo, in situ determination of amounts of critical metabolic indicators in body fluids or tissues of human patients. Examples of such body fluids include the blood and fluids associated with the lymphatic and neurological systems of the body. Further specific examples involving the human circulatory system include the monitoring of glucose and of oxygenated/de-oxygenated, arterial/venous colored hemoglobin in the blood stream. In addition, monitoring in localized tissue, such as brain and muscle, of certain enzyme species such as the cytochrome c oxidase enzyme (unofficially better known as cytochrome a, a.sub.3) or metabolic substrates (such as glucose) or products (such as carbon dioxide) is becoming an increasingly urgent practical application of spectrophotometric technology.
Spectrophotometric methods have been proposed in the art to monitor metabolites in corporeal fluids. Such methods involve the impingement of radiation, typically in the visible or near-infrared region, onto the exterior body portion of the subject for transdermal and interior tissue penetration of the radiation, which is monitored as to its reflectance or transmission, at a wavelength condition at which the metabolite or other monitored component is selectively absorptive for the radiation. This technique is mainly limited to yielding a qualitative determination from the measured output radiation (reflected or transmitted) of the qualitative character of the metabolism. At best a semi-quantitative result can be obtained in a so-called trend monitoring mode where concentration changes can be monitored in terms of an original baseline condition of unknown concentration.
Solute concentrations in dilute fluid media can be theoretically quantified by the Beer-Lambert law, i.e., EQU log (I.sub.o /I)=d.times.E.times.c
wherein:
I.sub.0 =intensity of source radiation impinged on the sample; PA1 I=intensity of radiation transmitted through the sample; PA1 E=absorption (extinction) coefficient of the solute species at the wavelength of the source radiation impinged on the sample; PA1 d=optical distance (travel pathlength of the radiation transmitted through the sample); and PA1 c=concentration of the solute (dilute component) in the solution sample.
Although the foregoing Beer-Lambert Law equation permits a ready determination of solute concentration to be made in in vitro or other non-corporeal discrete sample systems utilized for conventional spectrophotometric assays, such direct, quantitative measurement is not possible in the intact body, even though the influent radiation is penetrative of the body elements of the corporeal system, e.g., bones, musculature, organs, and the like, since the scattering of radiation during its passage through the corporeal system is extensive and highly variable in character. Such scattering not only adds an unknown loss of radiation to the required information regarding specific absorption but by multiple scattering it also lengthens to an unknown degree the path length of those photons eventually emerging from the body element. As a result, it has not been possible to determine in an in vivo situation what the effective path length, d, of the impinged radiation actually is, prior to measurement of the transmitted or reflected radiation derived therefrom. In consequence, the absolute quantitation of solute concentrations in corporeal systems has been severely adversely limited.
Faced with the alternatives of invasive and traumatic sampling of the corporeal fluids of interest, or spectrophotometric methods which realize only qualitative or at best semi-quantitative measurement of changes in tissue or body fluid solute concentrations, there is a substantial perceived need in the art for a non-invasive, in vivo method of quantitatively determining the concentration in corporeal solution of a solute in a body fluid solvent.
A similar need exists in numerous other fields in which absolute concentrations of dilute component species in fluid media would materially assist the characterization of the fluid system. An example is atmospheric monitoring of "acid rain", i.e., airborne acidic contaminants which have in recent years proliferated and been determined to cause widespread biospheric damage, including the defoliation of forest stocks and spoliation of natural bodies of water and other aqueous environments. It is anticipated that in coming years with the increasing severity of the acid rain problem, correspondingly greater scientific and legislative efforts will be focused on the monitoring of acid rain with a view to controlling and minimizing its adverse impacts. Determinations in the naturally existing medium such as turbid water and hazy or cloudy atmosphere will be a great boon for direct, effective and rapid monitoring of these environments.
In numerous other industrial and natural systems there is a need to quantitatively monitor solute species in an indirect manner not involving the time, effort, and cost of discrete sample collection, purification and analysis.
U.S. Pat. No. 4,281,645 to F. F. Jobsis describes a spectrophotometric system for monitoring cellular oxidative metabolism by non-invasively measuring in vivo changes in the steady state oxidation-reduction of cellular cytochromes together with changes in blood volume, the oxidation state of hemoglobin and the rate of blood flow in the brain, heart, kidneys, other organs, limbs, or other parts of a human or animal body.
The methodology described in the Jobsis patent involves transmitting near-infrared radiation in at least two different and periodically recurring wavelengths through the corporeal environment, and detecting and measuring the radiation intensity which emerges at another, distant point or on the opposite side of the body, for the monitoring of biochemical reactions, utilizing an approximation of the Beer-Lambert law. One of such wavelengths selected for the measurement is in a range for which oxidized cytochrome a, a.sub.3, is selectively highly absorptive. One or more reference signals are provided at corresponding wavelengths outside the peak of the cytochrome absorption band but preferably in close proximity to the measuring wavelength. The difference or ratio between the measuring and reference signals is determined and non-specific changes in the intensity of transmitted radiation not attributable to absorption by the cytochrome species are eliminated. Thus, the system of this patent produces an output signal representing the difference in or ratio of absorption of the measuring and reference wavelengths by the organ or other corporeal portion of the body as a function of the state of the metabolic activity in vivo, which may be converted to a signal providing a substantially continuous measure of such activity. A related spectrophotometric reflectance technique is disclosed in U.S. Pat. No. 4,223,680 to F. F. Jobsis.
U.S. Pat. No. 4,655,225 to C. Dahne et al discloses a spectrophotometric system for non-invasive determination of glucose concentration in body tissue. The system involves irradiation of the exterior body portion with an optical light whose transmittance or reflectance is collected at selected band wavelength values for the glucose absorption spectrum and at a selected band wavelength value for the absorption spectrum of background tissue containing no or insignificant amounts of glucose. The measuring and reference radiation collected is then converted into electrical signals and utilized to determine glucose concentrations.
It is an object of the present invention to provide an improved method and apparatus for indirectly quantitatively, spectrophotometrically determining the amounts of a dilute component by using a reference component in the environment of interest.
It is another object of the invention to provide such a method for non-invasive, in vivo quantitative determination of the concentration of a dilute solute component in a corporeal solvent environment.
Other objects and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims.